Fluorescent dyes are amongst the classes of molecules which are most broadly used and important in the labeling and detection of biomolecules. Such fluorescent dyes or labels may be classified into low molecular weight organic fluorescent dyes on the one hand and into medium to high molecular weight biomolecules, the fluorescent polypeptides or proteins on the other hand.
The sensitivity which may be achieved for example in an immunoassay using a fluorescent dye as a label largely depends on the number and efficacy of fluorescent molecules present on the specific binding partner used in the detection system, e.g. on an antibody used in an immunoassay. It is generally accepted, that there is rather a low optimal density of low molecular weight fluorophores which may be introduced into, for example, an antibody. This is due to the fact that over-labeling causes negative effects like high background staining, quenching of fluorescence and/or reduced binding of the antibody to its antigen.
Many attempts are known in the art which have lead to quite some improvement in fluorescence detection methods. One attempt has focused on the coupling of low molecular weight fluorescent molecules to biopolymers and thereafter coupling the such labeled biopolymers to a member of a biological binding pair, for example to an antibody. U.S. Pat. No. 4,169,137 discloses that primary-amine-containing poly-functional polymeric backbone reagents, for example polyethyleneimine or poly-L-lysine can be used to provide a biopolymer which is intensively labeled with a low molecular weight fluorescent dye, for example, with fluorescein isothiocyanate, (FITC).
Fluorescent dextran derivatives have been used for increasing fluorescence intensity, and numerous of such fluorescent dextran derivatives are commercially available. See “Handbook of Fluorescent Probes and Research Chemicals, 6th edition, R. P. Haugland, Molecular Probes, Inc., Eugene, Oreg. 97402, (1996). Fluorescent dextran derivatives consist of soluble dextrans (that is, of dextrans with a molecular weight of 10,000, 40,000, 70,000, 500,000, and 2,000,000 Daltons) conjugated with various fluorescent dyes such as fluorescein, dansyl, rhodamine, and Texas Red. The degrees of substitution in these fluorescent dextran derivatives are 1-2 dye molecules per dextrans of 10,000 Daltons, 2-4 dye molecules per dextran of 40,000 Daltons, 3-6 dye molecules per dextran of 70,000 Daltons, about 64 dye molecules per dextran of 500,000 Daltons, and about 134 dye molecules per dextran of 2,000,000 Daltons. Higher degrees of substitution tend to lead to fluorescence quenching and/or to non-specific interactions. Fluorescein isothiocyanate (FITC) derivatives of dextran or of poly-L-lysine with degrees of substitution ranging from 0.003 to 0.020 molecules of FITC per molecule of glucose and from 0.003 to 0.01 molecule of FITC per molecule of lysyl residue are commercially available from sources, such as Sigma Chemical Company.
Another attempt to increase fluorescence intensity used the fluorescent dye rhodamine, see Shechter, Y., et al., Proc. Natl. Acad. Sci., USA 75 (1978) 2135-2139. Higher than usual for fluorescence intensities were obtained for the peptide hormones insulin and epidermal growth factor by covalent attachment of these peptides to alpha-lactalbumin molecules that were highly substituted with rhodamine molecules (i.e., 7:1). This was accomplished while still retaining some binding affinity of each hormone for its receptor (which is one of the basic requirements of any labeling procedure).
Increasing the number of label molecules or particles per target site, however, does not always work. For example, H. M. Shapiro describes one attempt to increase fluorescence signals by Tomas Hirschfeld et al., at Block Engineering, wherein several hundred fluorescein molecules were attached to a synthetic polymer, polyethyleneimine, which was then conjugated with antibody. The method did not work because fluorescence emission from fluorescein molecules was quenched due to the short nearest neighbor distances between fluorophores on the same polymer molecule. See “Practical Flow Cytometry”, 3rd edition, H. M. Shapiro, Wiley-Liss, New York, N.Y., 1995, p. 277.
Although it obviously is possible to introduce a large number of low molecular weight fluorescent dyes into a desired conjugate, the biggest disadvantage even nowadays appears to be the fluorescence quenching which occurs if several low molecular fluorescent dye molecules are in too close a neighborhood.
An alternative attempt is to use latex particles which are labeled with low molecular weight fluorescent dye molecules. The advantages of such labeled microspheres are for example described in U.S. Pat. No. 5,516,635. Whereas such particles appear to provide for a better assay sensitivity, most likely due to a reduced fluorescence quenching, such particles in certain embodiments may suffer from disadvantages like difficulties in a reproducible production of such labeled latex particles, problems in stability, some leakage of fluorescent dye out of such particles and unwanted effects caused by the latex itself.
The medium to high molecular weight fluorescent polypeptides or proteins are quite different to the before discussed low molecular weight fluorescent dyes and therefore require different considerations and approaches. Such a fluorescent protein may carry up to about 30 chromophores per molecule. For example, it has been described that phycoerythrin (PE), a member of the phycobiliprotein family, may have as many as 34 associated chromophores. An antibody conjugated to PE may for example be used in a fluorescent plate immunoassay and such assay has been found to be quite sensitive (Custer, M. C., and Lotze, M. T., J. Immunol. Methods 128 (1990) 109-117).
Phycobiliproteins are light-harvesting proteins included in the phycobilisomes anchored in the thylakoid of cyanobacteria and red algae. They collect the visible light energy and channel it to the chlorophyll photosynthetic system. Due to their exceptional photophysical properties they are of great interest in fluorescence detection procedures.
The biliproteins are normally comprised of from 2 to 3 different subunits, where the subunits may range from about 10,000 to about 60,000 Dalton in molecular weight.
As mentioned above, phycobiliproteins will normally be comprised of 2 to 3 different subunits. For example, native APC consists of 6 phycobiliprotein subunits which make up the 104-kDa phycobiliprotein APC.
It is known that native APC dissociates into subunit monomers under most assay conditions (e.g., at a low protein and/or buffer concentration). This instability, e.g., in physiological buffers represents a major disadvantages of native APC.
It has been found, that APC preparations may be stabilized by intra-molecular cross-linking of APC. Cross-linked and stabilized allophycocyanin preparations (herein referred to as XL-APC) were developed by Glazer and Ong to make this dye more suitable for use in immunoassay (Ong, L. J., and Glazer, A. N., Physiol. Veg. 23 (1985) 777-787). Those authors took a standard preparation of APC and treated it with a chemical cross-linking agent, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDAC/EDC), such that an average of one alpha subunit was linked to one beta subunit in a covalent manner. The product was then denatured with 8 M urea to dissociate it into its component part: alpha and beta monomeric subunits and covalently linked alpha-beta dimeric subunits. The covalently-linked dimers were separated from the monomers using denaturing gel filtration, and then the dimers were placed in an environment that allowed them to re-associate into an (αβ)3 complex that displayed unusually high stability compared to native APC. The resultant material is referred to herein as XL-APC. This material has increased stability in the presence of chaotropic salts (such as sodium perchlorate) or at low concentration of buffer compared to native APC. When ran on a denaturing gel, most of the material runs as a single band that is the covalently linked αβ dimer. It is known that the higher the percentage of cross-linked αβ dimeric subunits, the better the utility of the XL-APC. A number of XL-APC preparations are commercially available that have various percentages of covalently stabilized αβ dimer all with greater than 50% of dimer in the final product.
Recently it has been shown (WO 01/96383) that a high fluorescent intensity APC may be obtained if the purification of such APC is performed under conditions avoiding the exposure of the biliproteins to strongly chaotropic agents. Such high fluorescent intensity cross-linked APC is reported to be about 28% more effective in terms of fluorescence quantum yield as compared to an APC as obtained by the methods previously applied. This APC (termed APC1) retains the same (αβ)3 trimeric structure as APC but incorporates a 10-kDa peptide linker in the core of the molecule. As for a standard APC it is also necessary to cross-link APC1 in order to produce a label of sufficient stability under routine assay conditions.
U.S. Pat. No. 5,891,741 discloses that it is possible to use aminodextran with a special activation characteristic to introduce up to about 20 PE molecules into such aminodextran. When labeled to an antibody of interest such PE-aminodextran leads to an increase in assay sensitivity in the range of about 2.2 to 5.6-fold. This increase in sensitivity goes to the expense of using aminodextran and therefore may also be subject to interferences caused by this carrier reagent. It also appears that for certain application the sensitivity as achieved by such method is not quite sufficient.
An alternative approach to overcome the problems encountered with respect to assay-sensitivity using purified phycobiliproteins has been the use of so-called phycobilisomes as described in EP 821 742. Phycobilisomes are complexes of phycobiliproteins and colorless polypeptides which function as the major light harvesting antennae in blue-green and red algae (Gantt, E., BioScience 25 (1975) 781-788). The major criterion for the functional integrity of these complexes is the demonstration that they exhibit highly efficient transfer of energy between component phycobiliproteins, for example, in Porphyridium cruentum phycobilisomes from phycoerythrin (PE) to phycocyanin (PC) and finally to allophycocyanin (APC). The colorless polypeptides are involved in the assembly and positioning of the phycobiliproteins within the phycobilisomes for proper stability and energy transfer.
Phycobilisomes from different organisms share a number of common properties, including: (1) extremely high “complex molecular weights” (5-20×106 Daltons) i.e., the weight of one mole of a phycobilisome complex comprised of multiple molecules; (2) multiple absorption maxima in the visible range of the electromagnetic spectrum; (3) high molar absorptivities (emax>107 M−1 cm−1); (4) efficient (>90%) directional vibrational energy transfer among constituent phycobiliproteins, commonly from one or more sensitizing species to a terminal acceptor capable of fluorescence; (5) large Stokes shifts relative to isolated phycobiliproteins; (6) high quantum yields of constituent phycobiliproteins; (7) high solubility in aqueous buffers; and (8) allophycocyanin-containing core structures.
Isolated phycobilisomes readily dissociate into free phycobiliproteins and a variety of phycobiliprotein complexes under all but the most favorable conditions. Like for purified APC measures have to be taken to stabilize phycobilisomes, e.g., by intra-molecular cross-linking.
As the skilled artisan will readily appreciate, phycobilisomes are difficult to purify and isolate and even more so to characterize, because of their variation in molecular weight as well as in polypeptide composition.
It therefore was a task of the present invention to provide for a method which when practiced leads to a fluorescent polypeptide complex which at east partially overcomes the above problems.